Method of preparing hydrated calcium phosphate gels



6 Claims. (Cl. 23-109) This application is a division of our'copendingapplication, Serial No. 699,951, filed December 2, 1957, now Patent2,967,802 of January 10, 1961, and is entitled to the filing datethereof.

This invention relates to a gel of a salt of an alkaline earth metal andto novel uses thereof.

It is an object of the present invention to prepare novel gels of saltsof alkaline earth metals.

A more specific object is to prepare a novel calcium phosphate gel.

A further object is to prepare novel immunological compositions, such asthose possessing antigenic properties.

Yet another object is to prepare novel pellet implants.

Still further objects and the entire scope of applicability of thepresent invention will become apparent from the detailed descriptiongiven hereinafter; it should be understood, however, that the detaileddescription and specific examples, while indicating preferredembodiments of the invention, are given by Way of illustration only,since various changes and modifications within the spirit and scope ofthe invention will become apparent to those skilled in the art from thisdetailed description.

According to the invention there is prepared a gel of a salt of analkaline earth metal. The gel so prepared has various uses; thus it hassorptive properties whereby it can be used to concentrate proteins andantigens. It can also be used as a tablet disintegrator, as a suspendingagent, as a clarifying agent, as an immunological adjuvant, as aflocculating agent and as an oral detoxifying antiacid.

The gels are prepared by admixing an acid solution of an alkaline earthmetal with an aqueous alkaline solution of phosphate in a colloidalstate. As the alkaline earth metal compounds there may be mentionedorganic and inorganic salts of magnesium, calcium, barium and strontiumcompletely dissolved in an acid medium.

Typical starting compounds include magnesium carbonate, calciumcarbonate, calcium chloride, magnesium chloride, barium chloride,strontium chloride, calcium acetate, calcium lactate, calcium gluconate,barium lactate, magnesium acetate; calcium propionate and calciumnitrate.

The preferred starting materials are the calcium salts. For best resultsin obtaining a pure compound calcium carbonate is then dissolved in anacid solution, e.g., lactic acid, hydrochloric acid, acetic acid, malicacid, fumaric acid, formic acid, nitric acid. At least the theoreticalamount of acid should be used to dissolve the calcium carbonate. Inorder to hasten the dissolution an excess of acid, e.g., 10%, ispreferably employed.

To the calcium or other alkaline earth metal salt there is then addedrapidly and with stirring aqueous ammonium phosphate in the presence ofexcess ammonia, e.g., suflicient to insure complete conversion of the"line thus reducing possibility of Cal-IP0 formation. The pH preferablyis not less than 9.1. This results in the formation of the gel. Sodiumhydroxide or potassium hydroxide may be employed in place of ammonia.

The preferred novel gel of the instant invention is pure hydratedcalcium phosphate of the formula.

Tribasic calcium phosphate as normally prepared contains a mixturerepresented by the formula together with adsorbed calcium and/orphosphate ions and contaminated with a variable quantity of CaHPO Thereis also present carbonates formed as a byproduct in the manufacture ofthe tribasic calcium phosphate. These impurities, and especially theCal-IP0 and carbonates markedly alter the physical and colloidalproperties of the freshly manufactured material and it is due to theirabsence that the hydrated calcium phosphate of the instant invention hasits unusual and unique properties.

The over-all chemical equation for the synthesis of the gel of thepresent invention can be represented as follows:

For best results the calcium compound and the phosphate should be usedsubstantially in the amounts indicated by the equation, namely, 10 molsof calcium to 6 mols of phosphate.

Example 1 The product has an afiinity for many organic and/ orbiological materials, for example, proteins, dyestuffs, and nitrogenoussubstances in general. It can be used to concentrate antigens includingboth proteantigens and other antigens.

(1) One thousand grams (l0 mols) of pure calcium carbonate weredissolved in 2350 grams of lactic acid (22 mols, i.e., theoreticalquantity+11 0%) (U.S.P.) in 7.5 gallons of distilled water. The carbondioxide formed was removed by boiling and the solution was mixed well.

(2) In a separate vessel 690 grams (6 mols of anhydrous H PO ofphosphoric acid (NR 85%) was dissolved in 7.5 gallons of distilledwater. There was added 1790 cc. of aqueous ammonium hydroxide solution(28 and the combination was well mixed. pH at this step is approximately9.1. Eighteen mols of ammonia are required to convert the 6 mols ofphosphoric acid to triammonium phosphate and the excess ammonia isneeded to insure complete formation of P0, with a minimum of HPO,,.

(3) Then the basic ammonium phosphate solution was added rapidly withrapid stirring to the calcium lactate solution, or admixedsimultaneously, and the re sulting suspension allowed to standovernight, or for 18 to 24 hours.

The supernatant liquid was decanted and the suspension filteredmechanically. The cake was washed four times with 7 to 10 gallons ofdistilled water. The final wash water was ammonia free as shown byNesslers reagent. The cake was not allowed to dry during washing. Thenthe cake was placed in a calibrated container and brought to 5 gallonswith distilled water and mixed well.

The abov procedure produced a gel containing 935 grams dried solids(4.675% solids) and amounted to a yield of 93.5%. The gel had an acidcombining power 3 per gram of ignited material of 11.36 cc. of N/l HCl;an ash of 4.37% and loss on ignition of 6.5%.

The equations for the reactions in steps 1, 2 and 3 are as follows:

H26 100800 2OCH3CHOHGOOH 10Ca 0 O C CHHCH3)2.5H20

HQPO4 18NH4GH 6(NH4)3PO4 1815120 (3) 10Ca(OOCCI-IOHCH 51-1 06 (NI-I POIf the gel is to be stored for 3 days or longer 4% formalin solutionshould be added as a preservative. In place of formalin otherpreservative such as phenol or chlorobutanol can be used.

The concentration of solids in the gel can be varied. The gel cancontain as much as 15% solids but i usually more dilute.

For use as a protein concentrating medium the solids are normallydiluted from 2 to In a specific example of use as a proteinconcentrating medium the following procedure was employed:

Example 2 Media of proper concentration of nutrients specifically wasprepared and sterilized. The media was inoculated with inoculumcontaining a pure culture of Erysipelothrix rhusiopathiae and incubatedat 37 C. for 48 hours. Formalin is added to kill organisms. An amount ofthe gel of Example 1 (4.675% suspension) was added equivalent toapproximately 15% of the total culture media volume. The mixture wasshaken for 30 minutes mechanically. The container with mixture wasallowed to stand 48 hours whereupon the gel settled, removing theantigenic substances. This supernatant liquid was then decanted. Thefinal product was homogenized and tested for potency and proved to besatisfactory in every way.

The gel likewise can be dried and admixed with other materials normallyemployed in tablet formulations and compressed into tablet form. Thedried gel exhibits the property of absorbing water with swelling whichin turn causes disintegration or breaking of the tablet when takeninternally or used parenterally. The gel is dried for 24 to 48 hours (orto constant weight) at 110-130 F.

A typical tablet formulation is:

Gel (of Example I dried to constant weight at 20 c. Granulated with 1gm. starch paste.

The gel can be used as an adjuvant and concentrating medium in theproduction of immunizing agents including vaccines or bacterins, such asErysipelathrix rhusiopathiae, etc. These materials usually are primarilyproteins but they need not be so. Media of proper formulations, e.g.,agar, sugars, nutrient salts, meat extracts, etc., is inoculated withviable organisms and allowed to grow at the proper incubationtemperatures for an optimum period to produce antigenicity in knownmanner. When growth has taken place, e.g., 24 to 48 hours, apreservative sterlizing agent, e.g., formalin or phenol is added to killthe live organisms. A prescribed amount of gel, e.g., normally 2% to 5%solids concentration, preferably 15 of a 3% solids concentration, isadded to the media and thoroughly mixed or shaken to provide intimatecontact between the gel and the antigenic materials. The gel is allowedto settle to the proper degree for the concentration desired. Generally,final concentration is dependent upon potency desired. Concentration canalso be elfected by mechanical means such as filtration orcentrifugation,

To obtain the proper concentration supernatant liquid can be removed,added or adjusted to give the desired antigen strength. The product maybe homogenized if desired to insure uniform suspension.

The gels are particularly eifective in preparing pellet implants orsuspensions whereby the immunizing agent will prolong immunity byproviding longer contact between the antigen and tissue into which it isinjected or implanted.

A slow release of the antigen from the pellet or suspension is obtainedwhich results in the prolonged immunity. This slow release isaccomplished since the pellets formed have only a very Slight surfacesolubility. In suspensions, by its sorptive action, the gel will provideslow release of sorbed material, this prolonging action.

The antigen is concentrated with the tricalcium phosphate gel (or othergel) in the manner previously described. The concentrate of the gel andantigen is then placed in pans and lyophilized (dehydrated by freezedrying). The dried antigen is then combined with other materials such ascalcium sulfate, shell-ac, aluminum monostearate, magnesium stearate, orother materials normally used in tablet manufacture, granulated andprocessed into pellets or implants.

Alternatively, the antigen can be dehydrated by lyophilization withoutthe gel and the gel can be dried separately and then admixed with theantigen. Then other sorptive agents or fillers can be added if desiredand the mixture granulated and compressed into pellets.

Other formulas are as follows:

Example B In Example A the 8 gms. of calcium sulfate is replaced by amixture of 4 gms. calcium carbonate and 4 gms. calcium sulfate.

Example C In Example A the 8 gms. of calcium sulfate is replaced byanother excipient, namely, a mixture of 4 gms. lactose and 4 gms.calcium sulfate.

Other materials can be used alone or in combination with the gel toproduce a pellet which has only a very slight surface solubility. Suchmaterials include calcium carbonate, calcium sulfate, milk sugar,calcium oxide, etc. Preferably, however, the gel is included. The ratiosof the materials will vary with the amount of antigen and the degree ofhardness and insolubility desired.

Example D Gms. Erysipelas antigen (dried) 4.0 Calcium carbonate 2.0Calcium sulfate 1.0 Shellac (25% solution in alcohol) 2.0 Aluminummonostearate 0.5

The composition was granulated and compressed into pellets.

Example E Gms. Erysipelas antigen (dried) .6 Calcium carbonate 1 Calciumsulfate 8 Aluminum monostearate l Shellac 4 The composition wasgranulated and compressed into pellets.

The tricalcium phosphate gel is also an excellent suspending agent andvehicle for inclusion in suspensiontype products where it isadvantageous to include insoluble solids among the active ingredients,and also has antiacid properties which make it a vehicle as well as anactive ingredient in scour products for veterinary use andstomachic-intestinal products for human or veterinary use.

Typical scour formulations are:

Combination of sulfa drugs, kaolin, pectin, sodium thiosulfate, etc., inan aqueous suspension of gel in 2% to 14% concentration, depending uponquantities of solids present.

Typical stomachic-intestinal formulations are:

Grains Calcium phosphate gel of Example 1 6 Magnesium trisilicate 9containing aromatics, flavoring agents in aqueous suspension.

Unless otherwise stated, all parts and percentages are by Weight.

We claim:

1. A method of preparing a hydrated calcium phosphate gel free ofCal-IP0 comprising admixing an aqueous acid solution of a calcium saltwith an aqueous alkaline solution of a phosphate, the ratio of thecalcium ions to phosphate ions being approximately 10 to 6, the alkalinesolution being of sufiicient strength that after mixing with said acidsolution the over-all pH is at least about 9, and recovering said gel.

2. A method of preparing a hydrated calcium phosphate gel free of CaHPOcomprising admixing an aqueous acid solution of a calcium salt withaqueous ammonium phosphate in the presence of suificient excess ammoniato render the mixture alkaline to a pH of at least about 9, the ratio ofcalcium ions to phosphate ions being approximately 10 to 6, andrecovering the hydrated calcium phosphate gel.

3. A method of preparing a hydrated calcium phosphate gel free of CaHPOcomprising dissolving calcium carbonate in aqueous acid solution,removing the carbon dioxide formed and adding ammonium phosphate in thepresence of sufiicient excess of ammonia that the pH is at least about9, the ratio of the calcium ions to phosphate ions being approximately10 to 6, and recovering the hydrated calcium phosphate gel.

4. A method according to claim 3 wherein the acid selected from thegroup consisting of lactic acid, acetic acid, malic acid and fumaricacid is an organic acid.

5. A method according to claim 4 wherein the organic acid is lacticacid.

6. A method of preparing a hydrated calcium phosphate gel free ofCal-IP0 comprising dissolving calciurn carbonate in aqueous lactic acidsolution, boiling the solution to remove the carbon dioxide formed,adding a mixture of phosphoric acid and ammonium hydroxide having a pHof about 9 with stirring, the ratio of calcium carbonate to phosphoricacid being approximately 10 to 6, allowing the suspension to stand,removing the excess liquid to form a cake and washing the wet cake withwater until ammonia free, and recovernig the hydrated calcium phosphategel.

Plusje et a1 June 5, 1951 Marcus July 29, 1952

1. A METHOD OF PREPARING A HYDRATED CALCIUM PHOSPHATE GEL FREE OF CAHPO4COMPRISING ADMIXING AN AQUEOUS ACID SOLUTION OF A CALCIUM SALT WITH ANAQUEOUS ALKALINE SOLUTION OF A PHOSPHATE, THE RATIO OF THE CALCIUM IONSTO PHOSPHATE IONS BEING APPROXIMATELY 10 TO 6, THE ALKALINE SOLUTIONBEING OF SUFFICIENT STRENGTH THAT AFTER MIXING WITH SAID ACID SOLUTIONTHE OVER-ALL PH IS AT LEAST ABOUT 9, AND RECOVERING SAID GEL.